Proteins are chemical compounds endowed with tiological functions. Proteins with the ability to catalyze chemicaI reactions are designated enzymes.
Many proteins, especially enzymes from microbial sources, have found an extensive use in a variety of industrial applications. Enzymes have a number of advantages over purely chemical processes. They are highly specific, and can efficiently catalyze reactions which might otherwise require extreme conditions of e.g. temperature, pressure, pH, etc.
The following list is intended as a non-exhaustive list of particular examples. For a more detailed treatment of these and other industrial enzymes, reference is made to Godfrey and Reichelt (1983).
Amylases, glucose isomerases, glucoamylases, isoamylases, invertases, pullulanases, etc. are used in the starch conversion technology which is applied in the preparation of a great variety of products (Van Beynum and Roels, 1985). PA1 Beta-galactosidases, catalases, chymosines, lipases etc. are used in the dairy industry. PA1 Cellulases find an application in waste treatment industry. PA1 Proteases are used in the detergent, the leather and baking industries. PA1 Pectic enzymes are applied in the fruit juice industry PA1 Glucose oxidase is used as an antioxidant in brewing and wine-making industries and in the food industry in general, and also finds applications in clinical diagnostics and analytical assays. PA1 Most industrial enzymes are used preferably at elevated temperatures so that contamination and viscosity are reduced while reaction rates are increased. However, at industrially preferred temperatures, proteins are bound to undergo inactivation. Therefore, industrial enzymes are often best derived from thermophilic microorganisms. PA1 pH considerations must take into account the pH optimum of specific enzymes, the stability of substrate and products, and conditions further imposed by upstream and/or downstream operations in the industrial process. PA1 Enzyme activators and coenzymes present a problem to the extent that they create additional costs e.g. for their elimination from the product during down-stream processing. PA1 Inhibitors are a problem in as much as they can be difficult to remove from the reaction mixture or may be even essential for the reaction (e.g. in the case of substrate inhibition). Inhibitors can act through a variety of mechanisms, one of which is chemical modification of the protein. PA1 1 Hydrophobic interactions, which are thought to be the most important contribution favoring the folded state in aqueous environment (Kauzmann, 1959; Privalov, 1979). PA1 2. Chain entropy, i.e. the number of conformational states, which is the most important single contribution favoring the unfolded state. PA1 3 Specific non-covalent interactions between protein atoms in the folded state, which at least compensate for similar interactions made with the solvent in the unfolded state. These are hydrogen bonds, Van der Waals interactions, and other electrostatic interactions between charges (ionised groups) and/or dipoles (Creighton, 1983). PA1 1. The residue to be replaced should be directly involved in electrostatic interactions, preferably in the interface between subunits an/or domains. PA1 2. The mutation should occur at a site that can sterically accommodate the amino acid residue that is introduced. PA1 3. The residue should occur at a site of low solvent accessibility and, preferably be part of an interface between subunits and/or domains.
For industrial purposes either whole microbial cells or purified enzymes may be used. Purified enzymes, although more costly, are preferred because they can generally convert a higher proportion of substrate, while displaying fewer side reactions.
The use of enzymes in industry, and of proteins in general, is still restricted in many cases. The greatest technical difficulty is the finding of suitable proteins which are stable under industrially desired conditions such as temperature, pH, requirements of activators, and/or the presence of inhibitors.
The use of proteins for therapeutic applications is rapidly increasing, mostly due to developments in recombinant DNA technology. Recombinant tissue Plasminogen Activator, human serum albumin, human growth hormone, interferons, and insulin for instance are already well known in this respect (Watson et al., 1983; Roskam, 1987). Other examples are L-Asparaginase which finds an application in the treatment of acute lymphocytic leukemia, and superoxide dismutases which have been proposed for the treatment of oxidative damage in a wide variety of applications.
Last, but not least, a large variety of proteins are used for clinical or analytical assays (Bergmeyer, 1983), or as diagnostics (Dodet, 1987).
In all these applications where particular proteins are used, one is often confronted with the problem of protein stability Indeed, many proteins, particular enzymes, become unstable and inactivated when they are isolated from their natural environment.
Proteins commonly consist of combinations of some or all of twenty monomeric building blocks called amino acids. The amino acids are conventionally represented by either a three-or a one-letter code as shown in Table I.
The amino acids are linearly linked together by a special type of covalent bond termed the peptide bond. The order or sequence of the amino acids so linked is the primary structure of a protein. The primary structure is important for at least three reasons. First, it determines the three-dimensional (3D) structure of a protein. Second, it confers the latter three-dimensional conformation with one or more biological functions via the choice of amino acids necessary for the conformational structure yet bestowed with the appropriate chemical physical characteristics to fulfill the desired functionality. Third, essential information regarding the DNA sequence can be ascertained from the primary structure of a protein and vice versa.
A polypeptide is the amino acid chain that is translated from a single messenger RNA (mRNA) which, in turn, is transcribed from a single structural gene. Proteins are composed of one or more polypeptide chains. Monomeric proteins are composed of a single polypeptide chain (and in some cases, e.g. insulin, of a few covalently linked polypeptide chains). Oligomeric proteins are composed of two or more polypeptide chains, termed "subunits", which are structurally similar to monomeric proteins without necessarily possessing separate biological functions.
The three-dimensional structure of a protein comprises its secondary, tertiary, and quaternary structures. Secondary structure refers to structural elements that involve the relationship or interaction of the main-chain atoms of neighboring amino acids of the primary chain. Tertiary structure is the spatial arrangement of all atoms of the amino acids making the polypeptide chain. Many proteins may associate into super-assembly structures; in such multi-subunit proteins, the term quaternary Structure refers to the arrangement of subunits in the protein and quaternary interactions are those which occur between atoms belonging to different subunits.
Owing to secondary, tertiary, and quaternary structure, many complex interactions may occur between amino acids, whether they are close or far remote from each other within the amino acid sequence.
In the native state --i.e. the state associated with biological function--, the polypeptide chain adopts one, or a small number of, well defined conformations. In the denatured state, the protein is devoid of biological function; in this so-called "unfolded" state, the three-dimensional structure is not well defined in that the polypeptide chain assumes a large number of different conformations (Anfinsen & Sheraga, 1975).
The native state is stable under well defined conditions of pH, temperature, ionic strength and pressure. Where unfolding --denaturation-- is shown to be reversible, it is verified that the free energy of the native state is lower than that of the denatured state. In such instance, the difference in free energy (delta G) between the two states provides a direct measure of protein stability (Privalov, 1979).
In addition to covalent interactions, the native conformation results from a complex interplay of different forces (Janin,1979):
Folding is the process that leads to the formation of the native state, whereas denaturation is the process which leads to the denatured (or unfolded) state starting from the native conformation.
The kinetics of the unfolding --or of the folding-- process is, under given physical conditions, a function of the energy barrier encountered by the system during the transition process (Creighton, 1983). It is not a function of the difference in free energy between the native and denatured states.
In multi-subunit (oligomeric) proteins, individual subunits often display an appreciable degree of folding prior to their assembly into the final quaternary structure. The structure of the isolated subunits, however, need not be absolutely identical to that observed in the final oligomeric state. Upon recognition and association, further structural adjustments may indeed occur so that the final quaternary conformation acquires its most thermodynamically favored state (Jeanicke, 1987).
In polypeptide chains above a certain size (about 100 amino acid residues), the presence of globular sub-structures, termed "domains", has been recognized. Based on the analysis of a number of protein 3D structures (see review by Janin & Wodak, 1983), as well as on experimental data on protein renaturation (Goldberg, 1969), and on the discovery that such domains could be isolated by limited proteolysis (Porter, 1973), it is believed that domains could play an essential role in folding (Wetlaufer, 1973).
Structural domains can be identified in proteins with known 3D structures using the procedure described by Wodak and Janin (1981). In case the structure of a protein is not known, indications of domains can be obtained by studies on limited proteolysis (Porter, 1973).
It has been inferred from the above considerations that interactions between subunits or between domains contribute to increase the stability of the folded native state (Miller et al., 1987), and that folded individual domains, or subunits, can be folding intermediates. The early steps in protein denaturation are thus likely to involve the disruption of interactions between subunits or domains (Ptitsyn, 1987).
Such contention is in general widely and largely accepted by those experts in the art as a result of numerous observations derived from the study of naturally occurring protein mutants (Perutz, 1978; Walker et al., 1980; Mrabet et al., 1986).
Enzymes are proteins which have the ability to catalyze biochemical reactions. Enzymes are very specific for their substrate, a consequence of the 3D structure and of the physical-chemical properties of their "active site" where the substrate binds and the chemical reaction takes place (Fersht, 1985).
The partial or total loss of enzymatic activity (or of biological activity in proteins, in general) is termed inactivation. As a result of the above-mentioned considerations, any alteration of the native conformation of the enzyme may influence the enzymatic activity, and, in particular, lead to inactivation.
Thermal inactivation of enzymes, i.e. the loss of enzymatic activity as induced by temperature, may be either reversible or irreversible, depending on whether return to ambient temperature results in the recovery of biological activity within a reasonable period of time (Klibanov, 1983). The most common causes of irreversible denaturation are believed to be either non-covalent (aggregation or folding into a stable non-native conformation) or covalent (chemical modification of the covalent structure of the polypeptide chain). The latter has been shown to prevail in lysozyme (Ahern & Klibanov, 1985). A number of chemical reactions can take place in proteins at high temperature (Zale & Klibanov, 1986), but the most frequently studied ones are deamidation (Asn and Gln are converted to Asp and Glu, respectively), hydrolysis (e.g. cleavage of the acid-labile peptide bond following an Asp residue (Inglis, 1983), and oxidation of methionine to the sulfoxide form.
Covalent alterations of the protein structure --in particular, the introduction of chemical cross-links-- can also impart protein stability. It has indeed been the most common means for increasing protein (thermo)stability in the past (Torchilin, 1983; Sadana & Henley, 1986; Gottschalk & Jaenicke, 1987). The success of this approach has however been limited because (1) the protocols rely on exhaustive screening of cross-linking agents of variable lengths and/or chemistry, (2) the chemical reactions cannot be reliably targeted to specific amino acid residues of the protein and may therefore occur at unwanted sites, such as those which may interfere with biological function.
More recently, with the advent of protein engineering, it has been possible, by site-directed mutagenesis (SDM) of the gene, to modify a given protein amino acid sequence at will, and thereby produce enzymes with improved properties (for reviews see Knowles, 1987, and Dill, 1987).
In some of the early examples, SDM has been used to engineer non-native disulfide bonds in proteins. Only in a limited number of cases, however, has this approach been successful (Perry & Wetzel, 1984; Wetzel, 1985; Sauer et al., 1986; Villafranca et al., 1983 and 1987; Wells & Powers, 1986; Pantoliano et al., 1987). Reasons for this modest success are reviewed and discussed by Wetzel (1987) and Creighton (1988).
A more stable variant of T4 phage lysozyme has also been obtained by substitutions of the type X for Gly and of the type Pro for X where X is any other amino acid, attributing that effect to the reduced conformational freedom of the mutant due to the presence of "stiffer" residues (Matthews et al., 1987).
SDM has been used recently to study hydrophobic stabilization of bacteriophage T4 lysozyme resulting in one variant with increased stability (Ile to Leu substitution at position 3 (Matsumura et al., 1988).
SDM has also been used to probe the nature of electrostatic interactions --hydrogen bonds and interactions between charged groups-- and their influence on the pH dependence (Russel et al., 1987) and enzyme kinetic parameters of subtilisin (wells et al., 1987). But their influence on protein stability has not been investigated by this technique, nor has a more stable enzyme been produced as a result of specific modulation of electrostatic interactions.
The role of non-covalent interactions between protein subunits and/or domains has seldom been assessed by means of SDM. In a first example, Casal et al. (1987) could show that substitution of asparagine at position 78 by aspartic acid in dimeric yeast triosephosphate isomerase produced more labile protein. This result has been rationalized on the basis that deamidation of asparagine into aspartic acid is found to occur in proteins at elevated temperature. In a second example, simultaneous replacement of asparagine residues in the same model protein at positions 14 and 78 by threonine and isoleucine, respectively, was found to result in improved thermostability (Ahern et al., 1987). The doubling of the half-life of the mutant enzyme was, however, accompanied by a two-fold reduction in catalytic activity.
Temperature-sensitive mutations in bacteriophage T4 lysozyme were found to occur at sites of low solvent accessibility in the folded protein (Alber et al., 1987).